CHARACTERIZATION OF BUMETANIDE TRANSPORT IN ISOLATED SKATE HEPATOCYTES

The uptake of [H-3]bumetanide was studied in isolated ''skate hepatocytes in an albumin-free elasmobranch Ringer solution and compared with the uptake of bile acids in the presence of other cholephilic organic anions. [H-3]bumetanide uptake was energy dependent, temperature sensitive, and exhibited saturation kinetics. In contrast to taurocholate and cholate, which are transported only by Na+-independent mechanisms, removal of Na+ reduced the maximal uptake rate (V(max)) for bumetanide from 404 +/- 80 to 230 +/- 47 pmol.mg-1.min-1 without a change in the apparent Michaelis constant (K(m)). The apparent K(m) for the Na+-dependent portion of bumetanide uptake was 58 +/- 24 muM, and V(max) was 151 +/- 38 pmol . min-1 . mg-1. Taurocholate (100 and 200 muM) inhibited Na+-independent bumetanide transport competitively but was a noncompetitive inhibitor for Na+-dependent bumetanide uptake. Furosemide (100 muM) and two bumetanide analogues, PF-3034 (500 muM) and PF-2203 (500 muM), preferentially inhibited the Na+-dependent bumetanide uptake system, whereas cholate (100 muM) and probenecid (100 muM) preferentially inhibited Na+-independent bumetanide transport. The sulfhydryl (SH) reagents N-ethylmaleimide, 2,2'-dithio-bis(5-nitropyridine), and p-chloromercuribenzenesulfonic acid (PCMBS) inhibited both bile acid and bumetanide uptake. Dithiothreitol (500 muM) completely reversed the PCMBS-induced inhibition of bumetanide uptake. These results indicate that bumetanide is transported into hepatocytes of the small skate, Raja erinacea, by both Na+-dependent and Na+-independent mechanisms; the latter is shared by bile acids and probably sulfobromophthalein and other organic anions. Their uptake requires free SH groups.